Tenocyte containing bioscaffolds and treatment using the same

ABSTRACT

The present invention relates to methods for preparing bio scaffolds useful in the repair of tears. More specifically, the invention relates to a method of treating rotator cuff tear in a mammalian subject in need thereof comprising the steps of: (i) selectively expanding tenocytes in vitro in culture medium comprising insulin or a functional derivative and a glucocorticoid or a glucocorticoid-like molecule to produce a culture of expanded tenocytes; (ii) seeding a bioscaffold with said expanded tenocytes to produce a tenocyte seeded bioscaffold; and (iii) implanting said tenocyte seeded bioscaffold proximal to the rotator cuff tear. The present invention also relates to a bioscaffold comprising cells, wherein more than 80% of said cells are tenocytes.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a U.S. national stage application of InternationalApplication No. PCT/AU2008/000583, filed Apr. 24, 2008, which claims thebenefit of Australian Patent Application No. 2008901451, filed Mar. 26,2008 and Australian Patent Application 2007902168, filed Apr. 24, 2007,all three of which are incorporated herein by reference in theirentirety.

FIELD

The present invention relates to methods for preparing bioscaffoldsuseful in the repair of tears. More specifically, the present inventionrelates to the repair of rotator cuff tears using bioscaffolds seededwith tenocytes.

INTRODUCTION

Rotator cuff tendon tear is a common presentation resulting frominjuries sustained from overhead activities (Briner et al., (1997)Sports Med. 24(1):65-71). Numerous studies have described differenttechniques for tendon repair. These include suture techniques (Gerber etal., (1999) J. Bone Joint Surg. Am. 81(9):1281-90), tendon to bonefixation (Burkhart et al., (2000) Arthroscopy. 16(7):82-90), tendonmobilizing and sliding techniques for retracted tears (Tauro at al.,(1999) Arthroscopy. 15(5):527-30) and use of various tissue or synthetictendon grafts (Amstutz at al. (1976) J. Biomed. Mater. Res.10(1):47-59).

While surgical repair usually achieves high levels of functionalimprovement and patient satisfaction in the early post-operative stage,there is significant morbidity associated with large and chronic tears(Hattrup, (1995) J. Shoulder Elbow Surg. 4(2):95-100). In addition, thesuccess rate of revision surgery for failed rotator cuff repair is verylow (Bigliani at al. (1992) J. Bone Joint Surg. Am. 74(10):1505-15).Consequently, there is a need in the art to develop better methods oftreating rotator cuff tear.

In an attempt to improve surgical outcomes bioscaffolds and syntheticimplants (see U.S. Pat. Nos. 4,668,233, 4,775,380, 5,352,463, 4,902,508,and EP 0 223 370) have been proposed in the treatment of rotator cufftear. These devices are intended to encourage the invasion of tissue onimplantation, in the hope that the tissue ingrowth, together with thedevice, will confer improved wound healing and functional improvement tothe patient. However, devices for the treatment of rotator cuff tearhave generally failed to show successful long-term results with failurecommonly occurring due to the invasion of non-functional fatty tissue,tenosynovitis, loosening or implant failure. Further, continuous loadingof the device and abrasion against joint tissues causes wear, creep andfatigue of the device and ultimately the device fails.

As such, the augmentation of surgical repair with devices such asbioscaffolds in rotator cuff tears has not been shown to improve theclinical outcome of patients (Iannotti, (1994) J. Am. Acad. Orthop.Surg. 2(2):87-95). As such, the current methods are sub-optimal andthere is a need to develop better treatments for the repair rotator cufftear.

SUMMARY

The inventors have now developed a novel cell-based tissue engineeringapproach to the treatment of rotator cuff tear comprising theimplantation of a bioscaffold that has been seeded with tenocytes whichhave been expanded in vitro.

Accordingly, in a first aspect the present invention provides a methodfor treating rotator cuff tear in a mammalian subject in need thereofcomprising the steps of: (i) selectively expanding tenocytes in vitro inculture medium comprising insulin or a functional derivative and aglucocorticoid or a glucocorticoid-like molecule to produce a culture ofexpanded tenocytes; (ii) seeding a bioscaffold with said expandedtenocytes to produce a tenocyte seeded bioscaffold; and (iii) implantingsaid tenocyte seeded bioscaffold proximal to a rotator cuff tear.

In some embodiments, the tenocyte seeded bioscaffold is cultured invitro for sufficient time to establish the tenocytes beforeimplantation.

In some embodiments, the rotator cuff tear is a massive rotator cufftear.

It will be appreciated that the tenocytes used in the methods anddevices of the invention as described herein can be isolated from anytenocyte-containing tissue. In some embodiments the tissue is a tendon.The tendon may be from any anatomical site of an animal and may be arotator cuff tendon, supraspinatus tendon, subcapularis tendon,pectroalis major tendon, peroneal tendon, achille's tendon, tibialisanterior tendon, anterior cruciate ligament, posterior cruciateligament, hamstring tendon, lateral ligament, medial ligament, patellatendon, biceps tendon, and triceps tendon.

In some embodiments, the tenocyte containing tissue may be isolated fromany mammalian animal including, but not limited to a sheep, a cow, a pigor a human. In other embodiments, the tenocyte containing tissue isisolated from a human. In still other embodiments the tenocytecontaining tissue is isolated from the subject in need of treatment.

The isolated tenocytes are selectively expanded by in vitro culture inthe presence of a culture medium comprising insulin or functionalderivative. In some embodiments the culture medium comprises about0.00005% to 0.1% w/v insulin or functional derivative. In otherembodiments the culture medium comprises about 0.0001% to 0.001% w/vinsulin or functional derivative. In still other embodiments the culturemedium comprises about 0.0006% w/v insulin or functional derivative.

The culture medium may further comprise a glucocorticoid, such as asynthetic glucocorticoid, or a glucocorticoid-like molecule. In someembodiments the glucocorticoid is betamethasone. The culture medium maycomprise about 0.00001% to 0.1% w/v glucocorticoid or aglucocorticoid-like molecule. In some embodiments the culture mediumcomprises about 0.0001% to 0.001% w/v glucocorticoid or aglucocorticoid-like molecule. In still other embodiments the culturemedium comprises about 0.0002% w/v glucocorticoid or aglucocorticoid-like molecule.

In some embodiments, the culture of expanded tenocytes comprises atleast 80% tenocytes and no more than 20% non-tenocyte cells. In otherembodiments at least 90% of the cells in the culture are tenocytes. Instill other embodiments at least 95% of the cells present in theselectively expanded tenocyte culture are tenocytes.

In some embodiments, the expanded tenocyte culture comprises cells,wherein at least 80% of said cells express one or more genes coding forthe following: type I collagen, type III collagen, EphA4, scleraxis,Six1, COMP and/or Cbfa1.

In a second aspect, the present invention provides a method of treatingmassive rotator cuff tear in a human subject in need thereof comprisingthe steps of: (i) isolating tenocytes from tendon tissue taken from saidsubject; (ii) selectively expanding said tenocytes in vitro in a culturemedium comprising about 0.0006% w/v insulin or functional derivative andabout 0.0002% w/v glucocorticoid or a glucocorticoid-like molecule toproduce a culture of expanded tenocytes comprising at least 80%tenocytes; (iii) seeding a bioscaffold with said expanded tenocytes andculturing said bioscaffold and tenocytes for no more than five days toproduce a tenocyte seeded bioscaffold; and (iv) implanting said tenocyteseeded bioscaffold into the rotator cuff tear.

In a third aspect, the invention provides a bioscaffold comprisingcells, wherein more than 800 of said cells are tenocytes. In someembodiments, the bioscaffold comprises at least 90%, 95% or 99% tenocytecells.

The bioscaffold may comprise cells, wherein at least 80% of said cellsexpress one or more genes coding for the following: type I collagen,type III collagen, EphA4, scleraxis, Six1, COMP and/or Cbfa1.

The bioscaffold may comprise a matrix, a membrane, a microbead, afleece, a thread, or a gel, and/or mixtures thereof. In some embodimentsthe bioscaffold comprises a type I/III collagen matrix (ACI Matrix™) orsmall intestinal submucosa (Vitrogen™).

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Experimental design of animal study.

FIG. 2: Model of massive rotator cuff tear in the rabbit.

FIG. 3: Expression of type I/III collagen and EphA4 in culturedtenocytes and tendon tissue.

FIG. 4: Immunostaining for type I collagen on tenocytes in vitroculture. (A) Test well (100×); (B) control well (100×).

FIG. 5: (A) The loose, porous collagen fibre arrangement of theACI-Maix™ bioscaffold (100×). (B) A sheet of tenocytes integrated withinthe ACI-Maix™ collagen fibres after 72 hour (200×). (C) Solo cellmorphology demonstrated two types of protrusions (blebs: black arrow &lamellipodia: white arrow) on the ACI-Maix™ bioscaffold (1500×). (D)Cross-sectional scanning electron microscopy revealed the flat surfacemorphology and highly compact collagen structure of the Restore™ smallintestine submucosa bioscaffold (400×). (E) Tenocytes (arrows) displayeda monolayer growth pattern on Restore™ bioscaffold similar to that seenin vitro, with a spindle-shaped appearance and bi- or tripolarlamellipodia cell attachment (200×). (F) An individual cell on theRestore™ bioscaffold with protrusion (blebs: black arrow & lamellipodia:white arrow) seen on the cell surface (650×).

FIG. 6: Control group; hematoxylin and eosin. (A) Four week controlsamples were characterized by an increase in cellularity andneoangiogenesis and alteration in the organization of collagen fibres(250×). (B) Bone trough (BT) of 4 week samples were characterized byincreased cell population and the absence of mineralized zone (100×).(C) Eight week samples displayed a more organized structure with lesscell density and uniformly arrayed parallel collagen fibre arrangement(100×). (D) Bone trough of 8 week samples demonstrated typical mature4-zone structure (100×). MT: Mid-substance of regenerated tendon.

FIG. 7: ACI-Maix™ bioscaffold group; hematoxylin and eosin. (A) Largeportions of the ACI-Maix™ bioscaffold were not absorbed at 4 weeks, withobvious lymphocyte infiltration surrounding the bioscaffold (25×). (B)The absorbed area of 4 week samples demonstrated primary repair patterncharacterized by rough collagen arrangement, increasing cellularity, andneoangiogenic formation (100×). (C) The bone trough of 4 week sampleswas dominated by repaired cartilage tissue, with dense fibrous tissueextending from the edge of the bone trough (100×). (D) Eight weeksamples displayed more organized structure (100×). (E) The bone troughformation was also mature. (F) Inductions of adipose tissue (arrows) inthe mid substance of the regenerated tendon (100×) U: unabsorbedbioscaffold; L: lymphocytes invasion; A: absorbed area V: vascularisedtissue.

FIG. 8: Restore™ bioscaffold group; hematoxylin and eosin. (A) Theoriginal structure of the Restore™ SIS bioscaffold was seen asdisorganized fibres. Numerous inflammatory cells were observed insideand around the implant (25×). (B) The absorbed area demonstratedincreasing fibroblast number with both round and spindle shapedmorphology present in a richly vascularised connective tissue matrix(100×). (C) An 8 week sample did not completely absorb and presentedobvious lymphocyte infiltration (100×). (D) Bone tendon junctionexhibited a mature 4-zone structure (100×). A: Absorbed area; U:unabsorbed bioscaffold; L: lymphocytes; V: vascularised tissue.

FIG. 9: ACI-Maix™ bioscaffold seeded with autologous tenocytes group;hematoxylin and eosin (A) At 4 weeks, ACI-Maix™ bioscaffold implantedwith tenocytes was not fully absorbed and elicited lymphocytic cellsinfiltration (100×). (B) Few implanted autologous tenocytes (arrows)were also spotted within the remaining bioscaffold (200×). (C) At8-weeks, ACI-Maix™ bioscaffold implanted with tenocytes displayedidentical result as control group (100×). (D) The bone trough (BT)histology was indistinguishable from the control group (100×). A:Absorbed area; U: unabsorbed bioscaffold; L: lymphocytes; MT: Midsubstance of regenerated tendon.

FIG. 10: Restore™ bioscaffold + autologous tenocytes group; hematoxylinand eosin. (A) At 8 weeks Restore™ bioscaffold implanted with autologoustenocytes demonstrated excellent tendon structure similar to controlgroup (100×). (B) Tendon bone junction was also mature, displayingtypical 4-zone structure (100×). (C) Some granulation tissue was spottedmixed with the collagen fibres, resembling chronic granuloma response(100×). (D) Small foci of ectopic bone were found in all groups thatRestore™ bioscaffold was implanted (100×).

FIG. 11: Inflammation Rate: The percentage of repair tendon areasoccupied by inflammatory cells.

FIG. 12: Histology Scores for ACI-Maix™ and Restore™ bioscaffoldsimplanted alone or seeded with autologous tenocytes at 4 weeks and 8weeks.

FIG. 13: Immunostaining of ACI-Maix™+tenocytes for type I collagen (A)Test (blue arrow: cytoplasm area; black arrow: extra cellular matrix)(400×). (B) Negative control (400×).

FIG. 14: Type I collagen positive cells in ACI-Maix™ and Restore™bioscaffolds implanted alone or seeded with autologous tenocytes at 4weeks and 8 weeks.

FIG. 15: Working conditions for, and primer sequences of, rabbit type Icollagen sense primer (SEQ ID NO:1), rabbit type I collagen antisenseprimer (SEQ ID NO:2), rabbit type III collagen sense primer (SEQ IDNO:3), rabbit type III collagen antisense primer (SEQ ID NO:4), rabbitEphA4 sense primer (SEQ ID NO:5), rabbit EphA4 antisense primer (SEQ IDNO:6), rabbit GAPDH sense primer (SEQ ID NO:7), and rabbit GAPDHantisense primer (SEQ ID NO:8).

FIG. 16: Geometric measurement: Normal thickness, width and length weremeasured from fifty normal rabbit rotator cuff tendon harvested fromunoperated shoulder. All measurements were performed under slackcondition at room temperature.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to particularlyexemplified methods and may, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments of the invention only, and is notintended to be limiting which will be limited only by the appendedclaims.

All publications, patents and patent applications cited herein, whethersupra or infra, are hereby incorporated by reference in their entirety.However, publications mentioned herein are cited for the purpose ofdescribing and disclosing the protocols and reagents which are reportedin the publications and which might be used in connection with theinvention. Nothing herein is to be construed as an admission that theinvention is not entitled to antedate such disclosure by virtue of priorinvention.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of cell culture, cell biology andorthopedic surgery, which are within the skill of the art. Suchtechniques are described in the literature. See, for example, Coligan etal., 1999 “Current protocols in Protein Science” Volume I and II (JohnWiley & Sons Inc.); Ross et al., 1995 “Histology: Text and Atlas”,3^(rd) Ed., (Williams & Wilkins); Kruse & Patterson (eds.) 1977 “TissueCulture” (Academic Press); Canale (ed.) 2003 “Campbell's OperativeOrthopaedics” 10^(th) ed. (St. Louis, Mo.: MD Consult LLC); and Albertset al. 2000 “Molecular Biology of the Cell” (Garland Science).

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural reference unless thecontext clearly dictates otherwise. Thus, for example, a reference to “acell” includes a plurality of such cells, and a reference to “an agent”is a reference to one or more agents, and so forth. Unless definedotherwise, all technical and scientific terms used herein have the samemeanings as commonly understood by one of ordinary skill in the art towhich this invention belongs.

Although any materials and methods similar or equivalent to thosedescribed herein can be used to practice or test the present invention,the preferred materials and methods are now described.

In some embodiments, the present invention is directed towards a methodfor treating rotator cuff tear.

The terms “treating” or “treatment” or grammatical equivalents thereofare used herein to cover the treatment of a rotator cuff tear in amammalian subject, preferably a human, and includes: (a) relieving orameliorating the symptoms of a rotator cuff tear, i.e., cause regressionof the symptoms of a rotator cuff tear; or (b) preventing or inhibitingrotator cuff tear from re-occurring in a subject that may be predisposedto rotator cuff tear, but has not yet been diagnosed as having it, e.g.a person who has previously been treated for rotator cuff tear. Theeffect of the treatment may be therapeutic in terms of a partial orcomplete cure of a rotator cuff tear or prophylactic in terms ofcompletely or partially preventing the re-occurrence of rotator cufftear.

The rotator cuff refers to the group of muscles and their tendons thatact to stabilize the shoulder. The rotator cuff muscles are a group offour muscles that surround the shoulder (supraspinatus, infraspinatus,teres minor and subscapularis). The four rotator cuff muscle tendonscombine to form a broad, conjoined tendon, called the rotator cufftendon, and insert onto the bone of the humeral head in the shoulder.

The term “rotator cuff tear(s)” refers to a tear of one, or more, of thefour tendons of the rotator cuff muscles or the conjoined tendon of therotator cuff. Tears of the rotator cuff tendon may be partial thicknesstears, full thickness tears or full thickness tears with completedetachment of the tendons from bone. Partial thickness tears refer tofraying of an intact tendon. Full thickness tears refer to wounds thatpersist through the entire tendon. These may vary in size from verysmall or pin-point to very large involving the majority of the tendon.Full thickness tears may also involve complete detachment of thetendon(s) from the humeral head.

In some embodiments, the rotator cuff tear is a massive rotator cufftear.

The term “massive rotator cuff tear” refers to tears of more than about5 cm and involves more than one tendon.

It will be appreciated that most mammalian animals comprise tendons andthat all these tendons comprise collagen fibres embedded in aglycoprotein-rich matrix. Thus, in the present specification the term“mammalian subject” refers to any mammal, such as a human or mammals ofeconomical importance and/or social importance to humans, for instance,carnivores other than humans (such as cats and dogs). Also provided foris the repair of rotator cuff injury in livestock, including, but notlimited to, domesticated swine (pigs and hogs), ruminants, horses, andthe like. The term does not denote a particular age. Thus, both adultand newborn subjects are intended to be covered.

In some embodiments, a first step involves the isolation of a populationof tenocytes from a source. The term “tenocyte” as used herein refers tothe spindle-shaped, fibroblast-like cells that are found in tendons ofall mammals. Tenocytes typically have elongated nuclei and thincytoplasm and are often found sitting on collagen fibres in tendons.Tenocytes can often be identified on the basis that they producecollagen type I and express the marker “scleraxis”. Accordingly, in someaspects the tenocyte cells of the invention are scleraxis expressingcells or cells capable of expressing collagen type I or scleraxis.

The term “source” as used herein refers to any tenocyte-containingtissue in any mammal. In some embodiments, the tenocyte-containingtissue is a tendon. A tendon is the tissue which connects muscle to bonein a mammal. The tendon may be from any anatomical site of an mammal andmay be a rotator cuff tendon, supraspinatus tendon, subcapularis tendon,pectroalis major tendon, peroneal tendon, achille's tendon, tibialisanterior tendon, anterior cruciate ligament, posterior cruciateligament, hamstring tendon, lateral ligament, medial ligament, patellatendon, biceps tendon, and triceps tendon.

Tenocyte cells may be isolated from a source in a variety of ways, allwhich are known to one skilled in the art. In some embodiments, tenocytecells can be isolated from a biopsy material by conventional methods. Asdescribed in more detail below, in some embodiments, tenocytes areisolated by enzymatic digestion.

In some embodiments, the tenocyte-containing tissue may be isolated fromany mammalian animal including, but not limited to a sheep, a cow, a pigor a human. In other embodiments, the tenocyte-containing tissue isisolated from a human.

In some embodiments, the tenocyte-containing tissue is “autologous”,i.e. isolated from the body of the subject in need of treatment forrotator cuff tear. For example, a mammalian subject with a rotator cufftear can have a biopsy taken from any tendon in their body. Such tendonsinclude, but are not limited to, tendon of flexor carpi radialis and thecalcaneus tendon.

Tenocyte cells may be obtained from biopsy material by appropriatetreatment of the tissue that is to serve as the source of the tenocytecells. Techniques for treatment of tissue to obtain tenocyte cells areknown to those skilled in the art see, for example, Freshney “Culture ofAnimal Cells. A Manual of Basic Technique” 2^(nd) ed. (A. R. Liss Inc.).For example, the tissue or organ can be mechanically disrupted and/ortreated with digestive enzymes or chelating agents to weaken theinteractions between cells making it possible to obtain a suspension ofindividual cells. Typically the method will include a combination ofmechanical disruption, enzyme treatment and chelating agents. In onetechnique the tissue is minced and treated simultaneously orsubsequently with any of a number of digestive enzymes either alone orin combination. Examples of enzymes useful in dissociating cellsinclude, but are not limited to, trypsin, chymotrypsin, collagenase,elastase, hyaluronidase, DNase, pronase, dispase, and the like. In someembodiments, enzyme compositions containing an aqueous mixture ofcollagenase having an activity of about 43 nkat/ml to about 51 nkat/ml,and chymopapain having an activity of about 0.22 nkat/ml to about 0.44nkat/ml are used for dissociating cells, such as described in U.S. Pat.No. 5,422,261. Mechanical disruption can also be accomplished by, forexample, the use of blenders, sieves, homogenizers, pressure cells, andthe like.

The resulting suspension of cells and cell clusters can be furtherdivided into populations of substantially homogenous cell types. Thiscan be accomplished using standard techniques for cell separationincluding, for example, positive selection methods (e.g., clonalexpansion and selection of specific cell types), negative selection(e.g., lysis of unwanted cells), separation based upon specific gravityin a density solution, differential adherence properties of the cells inthe mixed population, fluorescent activated cell sorting (FACS), and thelike. Other methods of selection and separation are known in the artsee, for example Freshney “Culture of Animal Cells. A Manual of BasicTechnique” 2^(nd) ed. (A. R. Liss Inc.).

In some embodiments, tendon tissues, which have been isolated by biopsy,are washed, dissected and digested to form explants which can be grownin cell culture to yield free tenocytes. In some embodiments, the biopsytissue is subjected to enzymatic digestion and/or subjected to agentssuch as ethylenediaminetetraacetic acid (EDTA) that bind or chelatesCa²⁺ on which cell-cell adhesion depends. Examples of enzymes suitablefor use include one or more of collagenase, trypsin, and proteases.

In some embodiments, minced tendon tissue of no larger than 1 mm isincubated in the presence of about 2.5% w/v trypsin and about 5.5% w/vcollagenase in standard tissue culture medium without phenol red for atleast 3 hours at about 37° C. in about 5% CO₂.

In some embodiments, after enzymatic digestion, the tenocytes arerecovered from the biopsy material through centrifugation of the biopsysolution, and washing the resulting pellet with cell growth medium.Alternatively, the tenocytes may be recovered from the biopsy solutionby filtration through, for example, a mesh such as a sterile 150 micronnylon mesh. Another approach is based on the tendency of some cell typesto adhere strongly to plastic or glass, which allows them to beseparated from components of a tendon which do not adhere as strongly.Alternatively, the cells may be separated from other components of thetendon using antibodies that specifically bind to the cell, for exampleusing antibodies conjugated to a matrix or coupled to a fluorescent dyewhich can then be separated by fluorescent-activated cell sorting(FACS). In some embodiments, the tenocytes are isolated by filtration ofthe biopsy solution through a 0.22 μm filter to remove matrix debris andthe filtrate centrifuged to form a cell pellet. The pellet is thenwashed in cell growth medium.

The isolated tenocytes are selectively expanded in vitro. The term“selectively expanded” as used herein refers to culturing the isolatedtenocytes in such a way as to grow and increase the number of tenocytecells to the detriment of other cell types.

In some embodiments, the isolated tenocytes are expanded in vitro in aculture medium comprising insulin or functional derivative. Insulin is ahormone which, in its naturally-occurring form, is produced by thepancreas. However, the insulin used in culture medium for the selectiveexpansion of tenocytes may be synthetic, such as recombinant insulin, ornaturally occurring.

A “functional derivative” of insulin is a molecule such as thosedescribed by Chan et al., 2000, “Insulin-through the ages: Phylogeny ofa growth promoting and metabolic regulatory hormone” (AmericanZoologist, 40(2):213-222) having the activity of insulin, namely, theability to culture tenocytes and includes biologically active fragments,variants, and derivatives of insulin.

In some embodiments, a “functional derivative” of insulin or a fragmentor variant thereof has one or several amino acid residues substituted bynaturally occurring or synthetic amino acid homologues of the 20standard amino acids. Examples of such homologues are 4-hydroxyproline,5-hydroxylysine, 3-methylhistidine, homoserine, ornithine, beta-alanineand 4-aminobutanoic acid, beta-alanine, norleucine, norvaline,hydroxyproline, thyroxine, gamma-amino butyric acid, homoserine,citrulline, and the like.

A functional derivative of insulin can be prepared using polyethyleneglycol (PEG) according to the method of Sehon and co-workers (Wie etal., supra) to produce an insulin molecule conjugated with PEG. Inaddition, PEG can be added during chemical synthesis of insulin. Othermethods of preparing a derivative of insulin or a fragment thereofinclude reduction/alkylation (Tarr, Methods of ProteinMicro-characterisation, J. E. Silver ed., Humana Press, Clifton N.J.155-194 (1986)); acylation (Tarr, supra); chemical coupling to anappropriate carrier (Mishell and Shiigi, eds, Selected Methods inCellular Immunology, W H Freeman, San Francisco, Calif. (1980), U.S.Pat. No. 4,939,239; or mild formalin treatment (Marsh, 1971, Int. Arch.of Allergy and Appl. Immunol., 41:199-215).

It should be noted that the term “functional derivative” does notinclude molecules such as insulin-like growth factor I or II.

The insulin or functional derivative may be incorporated into theculture medium prior to adding the tenocyte cells to be cultured.Alternatively, the insulin or functional derivative may be added to themedium throughout the culture, for example, by culturing the cells inthe presence of a cell feeder layer, such as beta cells, which secreteinsulin or a functional derivative.

As used herein a “fragment” is a portion of the insulin protein whichretains the function of insulin and in particular, the ability tosupport the growth of tenocyte cells in culture. A fragment of insulincan be at least about 10 amino acid residues in length, preferably about10-16 amino acid residues in length, and more preferably about 10-20amino acid residues in length.

A “variant” of insulin is an insulin molecule that has one or moresubstitutions such that the secondary conformation thereof remainsunchanged. Examples of such conservative substitutions include aminoacids having substantially the same hydrophobicity, size and charge asthe original amino acid residue. Such substitutions are generally wellknown to those skilled in the art of protein or peptide chemistry. Forexample, conservative substitutions include proline for glycine and viceversa; alanine or valine for glycine and vice versa; isoleucine forleucine and vice versa; histidine for lysine and vice versa; threoninefor cysteine and vice versa; glutamine for asparagine and vice versa;and arginine for glutamate and vice versa.

Another example of a variant of insulin is one in which the cysteineresidues have been substituted to minimise dimerisation via disulfidelinkages. Preferably the cysteine residues are substituted with alanine,serine, threonine, leucine or glutamic acid residues. In addition, aminoacid side chains of insulin or fragment or derivative thereof can bechemically modified. Another modification is cyclisation of the insulin.

In some embodiments the culture medium comprises about 0.00005% to 0.1%w/v insulin or functional derivative. In other embodiments the culturemedium comprises about 0.0001% to 0.001% w/v insulin or functionalderivative. In still other embodiments the culture medium comprisesabout 0.0006% w/v insulin or functional derivative.

The culture medium may further comprise a glucocorticoid, such as asynthetic glucocorticoid, or a glucocorticoid-like molecule.Glucocorticoids are a class of steroid hormones characterised by anability to bind to the cortisol receptor and trigger similar effects,such as affecting metabolism or anti-inflammatory or immunosuppressiveeffects. Glucocorticoids may be naturally-occurring (hormones) orsynthetic (drugs).

Examples of synthetic glucocorticoids suitable for use in culture mediumfor the selective expansion of tenocytes include hydrocortisone,cortisone acetate, predisone, prednisolone, methylprednisolone,dexamethasone, betamethasone, triamcinolone, beclometasone,fludrocortisones acetate, deoxycorticosterone acetate (DOCA), andaldosterone.

A “glucocorticoid-like” molecule may be any molecule having an activityof a glucocorticoid, namely the ability to culture tenocytes. Examplesof glucocorticoid-like molecules suitable for use in the inventioninclude the antihepatocarcinogen, Rotenone (Youssef et al., 2003, J.Carcinogenesis 2:2), Rifampcin (Calleja et al., 1998, Nat. Med.,4:92-96), Glycyrrhizin (a component of licorice) (Kuroyanagi & Sato,1966, Allergy, 15:67-75), and Withanolides (from the herb Withanthiasomnifera) (Grandhi et al., 1994, J. Ethnopharmacol., 44:131-135).

In some embodiments the glucocorticoid is beta-methasone. Beta-methasoneis a synthetic glucocorticoid having the formula C₂₂H₂₉FO₅.

The culture medium may comprise about 0.00001% to 0.1% w/vglucocorticoid or a glucocorticoid-like molecule. In some embodimentsthe culture medium comprises about 0.0001% to 0.001% w/v glucocorticoidor a glucocorticoid-like molecule. In still other embodiments theculture medium comprises about 0.0002% w/v glucocorticoid or aglucocorticoid-like molecule.

In some embodiments, the isolated tenocytes are cultured for about 3days to about five weeks, at about 37° C. in about 5% CO₂ atmosphere.The time period for cell culturing can, of course, vary.

The resultant culture of “expanded tenocytes” can be termed asubstantially pure culture of tenocytes. The term “substantially pure”as used herein means that the predominant cells in the culture aretenocytes and other contaminating cells such as fibroblasts,chondrocytes and the like are of a lesser number. In some embodiments,the expanded tenocytes are at least 80% tenocytes and no more than 20%non-tenocyte cells. In other embodiments at least 90% of the cells aretenocytes. In still other embodiments at least 95% of the cells presentin the expanded tenocyte culture are tenocytes.

In some embodiments, the expanded tenocyte culture comprises cells,wherein at least 80% of said cells express one or more genes coding forthe following: type I collagen, type III collagen, EphA4, scleraxis,Six1, COMP and/or Cbfa1.

The selectively expanded tenocytes can be seeded onto a bioscaffold. Theterm “bioscaffold” refers to any matrix or scaffold that is suitable fortenocyte or cell adherence with or without an adhesive. By way ofexample and not limitation, the bioscaffold can be in the form of amembrane, microbead, fleece, thread, or gel, and/or mixtures thereof.The bioscaffold can be made out of any material that has the physical ormechanical attributes required for implantation, such as acting as ahaemostatic barrier. A haemostatic barrier inhibits penetration ofadjunct cells and tissue into the treated defect area.

In some embodiments the bioscaffold is made of a semi-permeable materialwhich may include cross-linked or uncross-linked collagen, preferablytype I in combination with type III, or type II. The bioscaffold mayalso include polypeptides or proteins obtained from natural sources orby synthesis, such as hyaluronic acid, small intestine submucosaperitoneum, pericardium, polylactic acids and related acids, blood(i.e., which is a circulating tissue including a fluid portion (plasma)with suspended formed elements (red blood cells, white blood cells,platelets), or other material which is bioresorbable. Bioabsorbablepolymers, such as elastin, fibrin, laminin and fibronectin are alsouseful in the present invention. Support matrix or scaffold materials asdescribed in US Publication No. 20020173806, herein incorporated byreference in its entirety, are also useful in the present invention.

The bioscaffold is preferably initially (i.e., before contact with thecells to be transplanted) free of intact cells and is resorbable withinthe mammalian subject. The bioscaffold may have one or several surfaces,such as a porous surface, a dense surface, or a combination of both. Thebioscaffold may also include semi-permeable, impermeable, or fullypermeable surfaces. Support matrices having a porous surface aredescribed, for example, in U.S. Pat. No. 6,569,172, which isincorporated herein by reference in its entirety.

The bioscaffold may be autologous or allogeneic. In some embodiments, asuitable autologous support matrix is formed from blood, as exemplifiedin U.S. Pat. No. 6,368,298, issued to Berretta, et al. on Apr. 9, 2002,herein incorporated by reference in its entirety.

A suitable bioscaffold may be a solid, semi-solid, gel, or gel-likescaffold characterized by being able to hold a stable form for a periodof time to enable the adherence and/or growth of cells thereon, bothbefore transplant and after transplant, and to provide a system similarto the natural environment of the cells to optimize cell growth.Examples of suitable support matrices are disclosed in US PublicationNo. 20020173806, which is hereby incorporated by reference in itsentirety.

Additional examples of suitable bioscaffold for growth of tenocytesinclude Vitrogen™, a collagen-containing solution which gels to form acell-populated matrix, and the connective-tissue scaffolds of Hwang (USpatent application no. 20040267362), Kladaki at al (US patentapplication no. 20050177249), Giannetti (US patent application no.20040037812) and Binette et al (US patent application no. 20040078077);all of which are incorporated herein by reference.

The bioscaffold can be cut or formed into any regular or irregularshape. In some embodiments, the bioscaffold can be cut to correspond tothe shape of the tear. The bioscaffold can be flat, round and/orcylindrical in shape. The shape of the bioscaffold can also be mouldedto fit the shape of a particular tear. If the bioscaffold is a fibrousmaterial, or has the characteristics of a fibre, the support matrix canbe woven into a desired shape. Alternatively, the bioscaffold can be agel, gel-like, or non-woven material.

In some embodiments the bioscaffold is comprised of porcine-derived typeI/III collagen, for example, ACI Matrix™. In other embodiments thebioscaffold is comprised of small intestinal submucosa, for exampleRestore™.

The term “seeded” refers to bringing the tenocyte cells into contactwith a bioscaffold for a sufficient time prior to transplantation suchthat they adhere (with or without an adhesive) to the bioscaffold. Insome embodiments the cells are cultured with the bioscaffold overnightor more than a week. In some embodiments, the selectively expandedtenocytes are cultured in vitro with the bioscaffold for about five daysbefore use.

In some embodiments, uniform seeding is preferable. It is believed thatthe number of tenocyte cells seeded does not limit the final tissueproduced; however optimal seeding may increase the rate of generation.Optimal seeding amounts will depend on the specific culture conditions.In some embodiments, the bioscaffold is seeded with about 0.05 to about5 times the physiological cell density of a native tissue type, i.e., intendon. In another embodiment, the cell density can be less than about1×10⁶ to 1×10⁷ cells, or more, per cm², typically about 4×10⁶ cells percm². In some embodiments, the bioscaffold is seeded with about 3.5×10⁶selectively expanded tenocytes per cm².

It will be appreciated that the bioscaffold seeded with selectivelyexpanded tenocytes can be packaged and sold as a device. Accordingly, insome embodiments, the present invention provides a bioscaffold seededwith tenocytes. The device packaging might comprise a plastic platesealed with a sheet of sterile adhesive film, as exemplified in U.S.Pat. No. 5,842,573, herein incorporated by reference.

In some embodiments, the device is implanted at a site proximal to therotator cuff tear.

The terms “implanted” or “implantation” or grammatical equivalentsthereof are used herein to cover any act that introduces a bioscaffoldcontaining tenocytes into the rotator cuff of a subject.

The term “proximal” refers to a site within the rotator cuff such thattreatment at that site will cause regression of the symptoms of therotator cuff tear.

In some embodiments the tenocyte seeded bioscaffold is implanted intothe tear cells facing down onto the tear. In some embodiments, acovering patch serves to cover the defect to further preventinfiltration of undesired materials, such as fibroblasts or macrophages,from the surrounding environment. In some embodiments, the coveringpatch may be any of the support matrices described herein, and/or caninclude collagen (type I/III), hyaluronic acid, fibrin and polylacticacid. Preferably, the covering patch is cell-free and resorbable, andmay be semi-permeable.

The tenocyte seeded bioscaffold may be secured in place by anyconventional means known to those skilled in the art, e.g. suturing,suture anchors, bone fixation devices and bone screws. In someembodiments, the tenocyte seeded bioscaffold is sutured into position.

The present invention also provides a bioscaffold comprising cells,wherein more than 80% of said cells are tenocytes. In some embodiments,at least 90% of the cells in the bioscaffold are tenocytes. In stillother embodiments at least 95% of the cells present in the bioscaffoldare tenocytes.

Alternatively, the bioscaffold may comprise cells, wherein at least 80%of said cells express one or more genes coding for the following: type Icollagen, type III collagen, EphA4, scleraxis, Six1, COMP and/or Cbfa1.

The invention will now be further described by way of reference only tothe following non-limiting examples. It should be understood, however,that the examples following are illustrative only, and should not betaken in any way as a restriction on the generality of the inventiondescribed above. In particular, while the invention is described indetail in relation to massive rotator cuff tear, it will be clearlyunderstood that the findings herein are not limited to massive rotatorcuff tear per se, but also encompasses the lesser rotator cuff tendoninjuries described supra.

Example 1 Animals and Bioscaffolds

Fifty Albino New Zealand White (Oryctolagus cuniculus) rabbits between12 to 20 weeks old with body weight between 3-5 kg were used in thisstudy. All rabbits were bred from an out bred rabbit colony maintainedin the animal house facility of the University of Western Australia(Nedlands, Australia). Rabbits were fed ad libitum with rabbit/guineapig pellets, hay, and provided water ad libitum. Rabbits were held incages measuring 1.5 m wide, 0.75 m long, and 0.75 m high, with gridfloors to prevent pod dermatitis. All operative procedures and cageactivities were conducted under strict guidelines detailed by theNational Health and Medical Research Council (NHMRC, Can berra,Australia).

Porcine-derived type I/III collagen bioscaffold (ACI Maix™) was suppliedand manufactured by Matricel (Herzogenrath, Germany). The collagenbioscaffold is a white complex with two different sides: the rough sideand the smooth side. The rough side appears as cross linking fibres withpore size around 200 μm while the smooth side shows a compactarrangement of fibres (Willers et al. 2005). The mechanical property atbreaking point is about 14.6+2.4N/mm² (Chen J. M. Unpublished data fromprevious study). A bioscaffold of small intestinal submucosa (Restore™)was manufactured by Depuy (USA). The small intestinal submucosa (SIS)bioscaffold is a 1 mm thick, highly compacted material withsemitransparent appearance which consists of 10 sheets of individualsmall intestinal submucosas laminated together by vacuum drying. Bothsides of the bioscaffold are very smooth (Zheng et al. 2005). The highlycompacted structure of the SIS bioscaffold endues it with a very strongtensile strength around 75.6+6.3N/mm² at breaking point (Chen J. M.Unpublished data from previous study).

Example 2 Experimental Design

The fifty rabbits were randomly allocated into five groups of ten (FIG.1). The left rotator cuffs were fully dissected and reconstructed by oneof the five following methods: (1) Group A (Control treatment): Thetendon excised during defect creation was in situ reimplanted into thedefect immediately and sutured to the bone trough using 5-0 absorbablesutures; (2) Group B (ACI-Maix™): The defect was repaired by suturingACI-Maix™ collagen bioscaffold as an interposition graft to the nativetendon and bone trough borders; (3) Group C (ACI-Maix™ with autologoustenocytes): The cell-bioscaffold composite was used to repair the cuffdefect in an identical manner to Group B; (4) Group D (Restore™): Thecuff defect was repaired in an identical manner to Group B, but usingRestore™ as an interposition graft; and (5) Group E (Restore™ withautologous tenocytes): The tenocytes seeded Restore™ composite was usedto repair the cuff defect in an identical manner to Group C. In eachgroup, five of the ten rabbits were euthanized at four weeks, and theothers euthanized at eight weeks.

All statistical data are expressed as mean±standard deviation andcompared by ANOVA using statistic software SPSS (SPSS inc. USA).P-values less than 0.05 were considered significant.

Example 3 Harvest and Culture of Tenocytes

As shown in FIG. 2 tendon tissue of 3 mm diameter was obtained by biopsypunch from the rabbit patellar tendon and was washed in DMEM F-12 medium(GIBCO, Invitrogen, USA) supplemented with 10% fetal bovine serum (FBS),100 μ/ml penicillin and 100 μg/ml streptomycin (hereafter medium refersto the same contents). The tendon tissue was then dissected into 0.5 mmof diameter and digested with collagenase (100 UI/ml Gibco, Invitrogen,USA) over night at 37° C. After the digestion, the solution was filterthrough a 0.22 μm filter to remove matrix debris and the cells releasedfrom tissue were centrifuged into pellet at 2000 rpm for 8 min.Supernatant containing enzymes were discarded and the pellet wasresuspend in new medium. This washing process was repeated three times.

The cell pellet of resultant tenocytes was then resuspended in 5 ml ofmedium and placed into a culture flask at density between 10³ to 10⁴cells/ml containing culture medium. This culture medium containsDulbecco's Modified Eagle Medium (DMEM) (Invitrogen), 15% v/v fetalbovine serum, 0.0006% w/v insulin, 0.0002% w/v betamethasone, 0.5% w/vpenicillin, 0.5% w/v streptomycin, and 0.6% L-proline at pH 7.0. Thecells are then incubated under 5% CO₂ at 37° C. The culture mediumshould be changed every third day for 3 to 5 weeks until cells reach themaximum cell numbers at the fifth passage, typically 20×10⁷ cells. Forimplantation, tenocytes of passage two were loaded onto either ACI Maix™(rough side) or Restore™ bioscaffold at a density around 3.5×10⁶/cm² andcultured for up to 5 days to formulate cell seeded bioscaffolds prior toimplantation.

Example 4 Comparison of in vivo and in vitro mRNA Expression inTenocytes

To confirm whether tenocytes maintained their phenotype duringcultivation, the expression of type I/III collagen and EphA4 mRNA wasexamined. Total RNA samples were extracted from both tendon tissue andin vitro cultured tenocytes using RNeasy Mini Kit (QIAGEN Australia).RNA was reverse-transcribed into cDNA using RETROscript™ First-strandSynthesis Kit (Ambion USA). Semi-quantitative RT-PCR was performed togenerate and amplify type I/III collagen and EphA4 cDNA. The final PCRproducts were analysed by electrophoresis on 1.2% agarose gel andvisualized by UV transilluminator (IBI) and Polaroid film. Theexperiment was repeated four times to ensure the integrity of theresult. The PCR amplification was performed according to the conditionslisted in FIG. 15.

Example 5 Immunostaining for Type I Collagen Protein on Tenocytes InVitro Culture

To confirm whether tenocytes maintain their ability to produce type Icollagen in culture, immunostaining was conducted in the followingsequences: place culture passage two tenocytes in a 6-well plate; at 50%confluence fix with 4% paraformaldehyde for 10 min at room temperature;block endogenous peroxidase with 3% H₂O₂ for 5 minutes; rinse in 3changes of Tris buffer saline (TBS); permeabilize cells with 0.1%TritonX100 for 5 min; wash twice with 0.2% bovine serum albumin (BSASigma, USA)/phosphate buffered saline (PBS); incubated with eithercollagen I (1:3000 abcam, cat: 106017, UK) primer antibody dilute in0.2% BSA/PBS for 1 hr at room temperature (negative control treated withPBS at this step); wash 4 times with 0.2% BSA/PBS, 4 times PBS and 4times 0.2% BSA/PBS; incubated with second antibody using LSAB system(Dako, cat: K0675 Denmark); repeat the washing procedure; visualizedwith DAB kit (Dako, cat: K3468 Denmark) staining the protein brown.

Example 6 Morphological Characterization of Tenocyte-Seeded Bioscaffolds

To determine the behaviours of tenocytes on the bioscaffolds, themorphology of tenocyte-seeded bioscaffolds on days 1, 3 and 5 of culturewas studied by scanning electron microscopy (SEM). Samples were fixed in2.5% glutaraldehyde (TAAB, Reading, UK) for 7 days at room temperaturethen treated with tannic acid. Samples were rinsed in 0.2M cacodylatebuffer; post-fixed in 1% osmium tetroxide in cacodylate buffer for 60min at room temperature; washed in 3 changes of cacodylate buffer;placed in 1% tannic acid in 0.05 cacodylate buffer for 60 min at roomtemperature; washed in saline solution; stained 60 min in 0.5% uranylacetate in double-distilled water; rinsed in saline solution; placed in25%, 50%, 70%, 95% and absolute ethanol sequentially for 30 min each atroom temperature; and then washed twice in super dry ethanol for 30 mineach at room temperature. After critical point drying, samples weremounted and viewed using a Phillips XL30 scanning electron microscope.

Example 7 Operative Techniques

Fifty rabbits were anaesthetized by intramuscular injection of Ketamine(Parke-Davis, Auckland, NZ) and Xylazine (Troy Laboratories Australia).A longitudinal incision over the left shoulder (The right shoulder wasnot operated.) was made and surgical exposure of the rotator cuff tendonwas achieved by releasing a portion of the trapezius and deltoid musclesfrom the acromion. The whole rotator cuff tendon was fully excised fromthe tendon-bone insertion to the muscle-tendon junction creating arectangle defect of approximate 7×8 mm² in area (FIG. 2). Using a dentalburr, a bone trough 10 mm long, 2 mm wide, and 2 mm deep was prepared atthe greater tuberosity perpendicular to the tendon fibre direction. Twosmall drill holes, 0.5 mm in diameter, were made from the lateral aspectof the humerus into the bony trough. Then the defect was repaired usingthe method correspond to its group. Following tendon reconstruction, thewound was closed in layers and dressed, but the limb was not splinted.

Example 8 Sample Harvest and Geometric Evaluation

At both 4 and 8 weeks, rabbits were anaesthetized and sacrificed byintravenous injection of pentobarbitone. The humerus head, cuff tendonand part of the muscle were harvested from both shoulders (operated andunoperated). Under slack condition, the length and width of both theoperated and unoperated tendon were measured with a vernier caliper andthe thickness was measured with a micrometer. Samples were then fixed in4% paraformaldehyde. After fixation, the specimens were decalcified with10% formic acid, dehydrated, paraffin-embedded, cut to 5 μm sections andstained with hematoxylin and eosin and Alcian Blue.

Example 9 Histology Examination

Inflammation response to the implants was evaluated by the inflammationrate which was interpreted as the percentage area of the repair tendonoccupied by inflammatory cells. The whole tendon area and areas occupiedby inflammatory cells were measured using graphical software Image ProPlus 4.5 (Media cybernetics, USA). General histological examination wasperformed with hematoxylin and eosin and Alcian Blue staining. Eightparameters were semi-quantitatively assessed (Movin et al. 1997; Shalabiet al. 2002): (1) fibre structure, (2) fibre arrangement, (3) roundingof the nuclei, (4) inflammation, (5) increased vascularity, (6)bone-tendon junction, (7) biocompatibility (absorption rate of theimplanted material), and (8) glycosaminoglycan content (the intensity ofblue colour on Alcian Blue section). Zero to three points were allottedto each of these variables, with 0 being normal and 3 being maximallyabnormal. Therefore, a perfectly normal tendon would have a score 0, anda maximally abnormal tendon would score 24.

The inflammation parameter was converted from the inflammation rate,which less than 10% scored 0, 10-20% scored 1, 20-30% scored 2, and morethan 30% inflammatory cell infiltrate scored 3. The interpretation ofbone-tendon junction was based on bone trough formation. If the bonetrough presented typical tendon junction histology consisting of 4zones, (cortical bone, mineralized fibrocartilage, fibrocartilage andtendon) it scored 0. The absence of one of these zones resulted in ascore increase of 1, and a junction completely dissociated scored 3.This scoring system is a modification of Likert's grading (Movin et al.1997; Shalabi et al. 2002).

Example 10 Immunostaining of Type I Collagen on Repair Tendon

To determine the ratio of cells that synthesized type I collagenprotein, immunostaining was performed in following sequence: afterdewaxing and rehydration, slides were digested with 0.1% trypsin for 20min to retrieve the antigen; wash with water; blocked with endogenousperoxidase with 3% H₂O₂ for 5 min; washed with TBS three times for 5 mineach; blocked with 20% fetal bovine serum (FBS) for 30 min; incubatedwith type I collagen primer antibody (1:3000 abcam, cat: 106017, UK) for3 hrs at room temperature (the negative control was treated with PBS atthis step); washed in TBS three times for 5 min each; incubated with asecond antibody using LSAB system (Dako, cat: K0675 Denmark); thewashing procedure was repeated; visualized with DAB kit (Dako, cat:K3468 Denmark), which stained the positive cells brown; andcounterstained with haematoxylin.

Five high power views (400×) of each slide were randomly selected. Thetotal cell number and type I collagen positive cells of each view werecounted. The ratio was calculated y dividing the type I collagenpositive cell number by the total cell number. The average of the fiveviews was the final ratio of the slide.

Example 11 Experimental Design

Fifty rabbits were randomly allocated into five groups of ten (FIG. 1).The left rotator cuffs were fully dissected and reconstructed by one ofthe five methods. In situ reimplantation of dissected rotator cufftendon served as the control. Five rabbits from each group weresacrificed at 4 and 8 weeks post-operatively.

A schematic of the rabbit massive rotator cuff tear model, used in thisstudy, is depicted in FIG. 2. Autologous tenocytes enzyme digested frombiopsy harvested from patellar tendon were cultivated in vitro andseeded on both bioscaffolds (cell density: 3.5×10⁶/cm²). When thecell-bioscaffolds were ready, the rotator cuff tendon was fully excisedfrom the insertion point of the humeral tuberosity to the muscle-tendonjunction creating a defect around 7×8 mm². In the control group, thiswas reimplanted into the excision site. In the other four groups, theexcised tendon portion was discarded and replaced by ACI-Maix™ andRestore™ bioscaffolds, with or without autologous tenocytes.

Example 12 Protein and mRNA Expression of Type I/III and EphA4 inTenocytes

Using RT-PCR analyses, we compared the gene expression profiles of typeI/III collagen and EphA4 in tendon tissue and cultured tenocytes. Asshown in FIG. 3, tenocytes maintained their ability to express collagentypes I/III and EphA4 mRNA in vitro. The level of expression appearedhigher in mRNA samples extracted from the in vitro cultured tenocytesthan that from tendon tissue when compared to house keeping gene GAPDH.

To further confirm if in vitro tenocytes produce type I collagenprotein, immunohistochemical staining of type I protein was performed inthe cultured tenocytes. The result shows that type I collagen protein inthe cytoplasm of in vitro cultured tenocytes (FIG. 4A), while in thecontrol well no positive staining was seen (FIG. 4B).

Example 13 Morphological Assessment of Tenocyte-Seeded Bioscaffolds

Scanning electron microscope analysis of the ACI-Maix™ collagenbioscaffold indicated a loose, porous collagen fibre arrangement withpore size of approximately 200 μm (FIG. 5A). At 24 hrs tenocytes hadadhered to the collagen matrix and formed a sheet-like arrangement. By72 hrs, abundant tenocyte replication had occurred and cells coveredmost of the bioscaffold surface (FIG. 5B). Higher magnification (FIG.5C) of individual cell morphology demonstrated two types of protrusions,blebs and lamellipodia, radiating from the cell surface. The Restore™SIS bioscaffold was characterized by a flat surface topology and highlycompact structure (FIG. 5D). The growth pattern of tenocytes on theRestore™ bioscaffold was similar to that in culture flask, displaying aflattened fibroblastic-like appearance. Tenocyte replication wasrelatively slow with a monolayer growth pattern (FIG. 5E). Highermagnification revealed that Restore™-seeded tenocytes (FIG. 5F) also putout large numbers of protrusions on the surface of the cell similar tothat on ACI-Maix™ bioscaffold.

Example 14 Gross and Examination

Following surgery, all rabbits survived until scheduled euthanasia.There were no wound infections or any other complications observed. Alimping gait from the operative forelimb was observed during the firstweeks post surgery, and then normal gait returned at 2 weeks. Grossexamination revealed that no pull-off or failure of tendon repair wasobserved in any sample at either time'point. The mean thickness, widthand length of fifty normal rabbit rotator cuff tendon harvested fromunoperated shoulder was 2.5+0.3 mm, 6.6+0.5 mm and 8.8+0.5 mm,respectively (FIG. 16). Generally, the average thickness samples at 4weeks was significantly (p<0.01) thicker than normal tendon, whilesamples at 8 weeks were significantly (p<0.01) thinner than normaltendon. No difference in width was found between normal and repairtendon. Both 4 and 8 week repair tendons were significantly elongated(4-5.5 mm longer, p<0.01) compared to normal tendon (FIG. 16).

Example 15 Histological Assessment of Control Treatment

Autologous tendon repair at 4 weeks post-surgery was characterized by anincrease in cellularity, neoangiogenesis, and alterations in the normallongitudinal arrangement of the collagen fibres (FIG. 6A). The collagenbecame wavier than that of normal tendon with some disorganized areasand slightly rounded tenocyte nuclei. The tendon-bone junction at thebone trough was fibrocartilage-like with large spherical cartilage cellsfilling the space, similar to the normal tendon-bone junction exceptwith a dense cell population and an absence of mineralized osteoidmatrix (FIG. 4B). By 8 weeks, samples displayed a more organizedstructure with normal cell density and shape, and a healthy parallelarrangement of reparative collagen fibres (FIG. 6C). The bone trough ofthe 8 week sample exhibited a more mature 4-zone formation and betterintegration with the bone marrow (FIG. 6D).

Example 16 Histological Assessment of Bioscaffolds WITHOUT AutologousTenocytes

ACI-Maix™ group: At 4 weeks post surgery, all ACI-Maix™ grafts implantedwithout autologous tenocytes displayed large portions of the bioscaffoldthat were not absorbed. In areas where the original structure of thebioscaffold remained, there were large number of lymphocytic cells seenwithin and surrounding the remaining bioscaffold (FIG. 7A). The areaswhere the graft was absorbed demonstrated a primary repair patterncharacterized by roughly parallel collagen arrangement, increasedcellularity and vascularisation (FIG. 7B). The bone trough was dominatedby repaired cartilage tissue, with dense fibrous tissue extending fromthe edge of the bone trough (FIG. 7C). At 8 weeks, the implant sitedisplayed more bundles of parallel collagen fibres; containing lowdensity fibroblastic cells and reduced neoangiogenesis (FIG. 7D). Bonetrough formation also matured as collagen fibre organization wasremodelled and produced good integration with the cortical and cancellorbone (FIG. 7E). However, two out of the five samples still containedfragments of unabsorbed bioscaffold with obvious lymphocyticinfiltration. Plus, inductions of adipose tissue in the mid-substance ofthe regenerated tendon were found in all five 8 week samples (FIG. 7F).

Restore™ group: In all five 4 week samples, Restore™ implanted withoutautologous tenocytes were only partially absorbed. The originalstructure of the Restore™ was present as disorganized bioscaffolds alongthe structure of tendon. Numerous inflammatory cells, predominantlylymphocytes, were observed at the margins of the graft (FIG. 8A). A fewmacrophages, plasma, eosinophil and polymorphic cells were occasionallyspotted within the bioscaffold fibres. In areas where the Restore™ hadbeen absorbed, large numbers of fibroblasts and lymphocytes were presentwithin a richly vascularised connective tissue matrix resemblinggranulation tissue (FIG. 8B). The bone trough was occupied by fibroustissue mixed with chondrocytes with the mineralized zone virtuallyabsent in all samples.

At 8 weeks, four of the five Restore™ bioscaffold implants were totallyabsorbed. The one that wasn't completely absorbed presented obviouslymphocyte infiltration similar to 4 week samples (FIG. 8C). In theabsorbed area, the collagen fibres were aligned in a less wavy patternand angiogenesis was less intense. The tendon-bone junction exhibited amature 4-zone structure including cortical bone, mineralizedfibrocartilage; fibrocartilage and tendon (FIG. 8D). Some granulationtissue was spotted between the void of collagen fibres, resemblingchronic granuloma response. Unexpectedly, some ectopic bone formationwas found in the mid-substance of two samples.

Example 17 Histological Assessment of Bioscaffolds with AutologousTenocytes

ACI-Maix™ group: At 4 weeks post surgery, both the neo-generated tendonand bone trough of ACI-Maix™ bioscaffold implanted with autologoustenocytes were similar to 4 week bioscaffolds implanted withouttenocytes. Some portions of bioscaffold still remained and lymphocyticcells were again seen within and surrounding the bioscaffold (FIG. 9A).Implanted autologous tenocytes were also observed on the surface ofunabsorbed bioscaffold (FIG. 9B). In areas where bioscaffold absorbed,rough collagen arrangement, and vascular beds were observed similar toother experimental groups.

Interestingly, at 8 weeks post implantation, superior reparative resultswere evidenced, with tendon histology similar to controls treatment(p>0.05) (FIG. 9C). All implanted bioscaffolds were fully absorbed andreplaced by tendon tissue at 8 weeks. Collagen bundles werelongitudinally arranged and the bone trough histological appearance wasindistinguishable from the control histology (FIG. 9D). A slightlyincreased amount of fibroblasts were evenly distributed between bundlesin a typically flattened, thin, and wavy pattern. Only one out of fivesamples contained adipose tissue in the middle of neo-generated tendon.

Restore™ group: Restore™ implanted with autologous tenocytes displayed asimilar repair pattern to other groups at 4 weeks. Excellent reparativeresults were seen at 8 weeks, highlighted by histological appearancesimilar to control tendon repair, demonstrating longitudinally arrangedcollagen bundles with fibroblasts evenly distributed between the bundlesin a spindle-shaped pattern (FIG. 10A) and mature bone trough formation(FIG. 10B). No unabsorbed bioscaffold was found in all five samples andmost the bone trough formation demonstrated typical four zone structure.However, granulation tissue (FIG. 10C) was still observed in all 5samples and ectopic bone (FIG. 10D) was found in 2 samples.

Example 18 Semi-Quantitative Assessment of Repair Tendon Histology

Analysis of the inflammation rate to the bioscaffolds demonstrated thatthe inflammatory response was less intense in the bioscaffold-cellconstruct groups than in the bioscaffold-only (FIG. 11). At 4 weeks, theaverage inflammatory rate (the percentage of repair tendon areasoccupied by inflammatory cells) was 56% with ACI-Maix™, whilst theACI-Maix™+cell group was significantly (p<0.01) reduced to 26%. Asimilar phenomenon was observed in groups applying Restore™ bioscaffold,the inflammatory rate decreasing from 46% to 27% favouring autologoustenocytes implantation (p<0.05). However, when compared to the controls,the inflammatory rate in the four experimental groups was significantly(p<0.05) elevated, regardless with or without cell. At 8 weeks, thebioscaffold absorbed, and the inflammatory response of all groups wassignificantly (p<0.05) reduced compared to 4 weeks. In groups thatautologous tenocytes were implanted, the inflammatory rates were verysimilar (p>0.05) to that of the control group, whilst the inflammationobserved in bioscaffold-only group was still significantly (p<0.05)greater than controls.

General histology examination revealed that, all experimental groupshave poor histological scores compared to the positive control (p<0.01)at 4 weeks (FIG. 12). At 8 weeks, ACI-Maix™ bioscaffold implanted alonedisplayed significantly inferior histological score to the control(p<0.05), whilst seeded with autologous tenocytes it displayed muchbetter score identical to the control (p>0.05). However, the differencebetween these two groups (ACI-Maix™ with and without tenocytes) was notsignificant (p>0.05). Although the histology score of Restore™bioscaffold without tenocytes was worse than implanted with tenocytesand control, it still achieved an outcome not statistical significantdifferent from either of them (p>0.05).

Immunostaining demonstrated that both cytoplasm and extra-cellularmatrix were stained positive for type I collagen (FIG. 13A) while nopositive staining was seen in the negative control (FIG. 13B). At 4weeks, the average type I collagen positive cell ratio of the controlgroup was 53.4%, ACI-Maix™ was 38.4%, ACI-Maix™ seeded with tenocyteswas 58%, Restore™ was 36.2%, and Restore™ seeded with tenocytes was48.8%. At 8 weeks, the type I collagen positive cell ratio slightlyincreased in all groups with the control group achieving 63.2%,ACI-Maix™ achieving 50.60%, ACI-Maix™ seeded with tenocytes achieving61.2%, Restore™ achieving 51.80%, Restore™ seeded with tenocytesachieving 65%. At both time points, the positive rates of tenocytesseeded bioscaffolds were significantly higher than those withouttenocytes (p<0.05, FIG. 14) and similar to the control (p>0.05).

The invention claimed is:
 1. A method of treating rotator cuff tear in amammalian subject in need thereof comprising the steps of: (i)selectively expanding tenocytes in vitro in a culture medium comprising:(a) at least 10% v/v serum; (b) insulin; and (c) betamethasone toproduce a culture of expanded tenocytes; (ii) seeding a bioscaffold withsaid expanded tenocytes to produce a tenocyte seeded bioscaffold; and(iii) implanting said tenocyte seeded bioscaffold proximal to therotator cuff tear.
 2. The method of claim 1, wherein said rotator cufftear is a massive rotator cuff tear.
 3. The method of claim 1, whereinsaid tenocytes are isolated from a tenocyte-containing tissue beforeselective expansion.
 4. The method of claim 3, wherein saidtenocyte-containing tissue is a tendon.
 5. The method of claim 1,wherein said tenocytes are from a mammalian animal selected from thegroup consisting of a sheep, a cow, a pig and a human.
 6. The method ofclaim 5, wherein said mammalian animal is a human.
 7. The method ofclaim 1, wherein said tenocytes are autologous.
 8. The method of claim1, wherein said culture medium comprises about 0.0006% w/v insulin andabout 0.0002% w/v betamethasone.
 9. The method of claim 1, wherein saidculture of expanded tenocytes comprises at least 80% tenocytes.
 10. Themethod of claim 1, wherein said culture of expanded tenocytes comprisesat least 80% tenocyte cells that express one or more genes encoding thefollowing: type I collagen, type III collagen, EphA4, scleraxis, Six1,COMP and Cbfa1.
 11. The method of claim 1, wherein said tenocyte-seededbioscaffold is cultured in vitro for a sufficient time to establish thetenocytes before implantation.
 12. The method of claim 1, wherein saidmammalian subject is a human.
 13. The method of claim 1, wherein saidbioscaffold comprises a matrix, a membrane, a microbead, a fleece, athread, a gel, or mixtures thereof.
 14. The method of claim 1, whereinsaid bioscaffold comprises a type I/III collagen matrix or a smallintestinal submucosa.
 15. The method of claim 1, wherein the step ofseeding a bioscaffold with said expanded tenocytes to produce a tenocyteseeded bioscaffold is carried out at a cell density of 0.05 to 5 timesthe physiological cell density of native tendon.
 16. The method of claim15, wherein the cell density is at least 1×10⁶ to 1×10⁷ cells per cm².17. The method of claim 15, wherein the cell density is 4×10⁶ cells percm².
 18. The method of claim 1, wherein said culture medium comprises atleast 15% v/v serum.
 19. A method of treating massive rotator cuff tearin a human subject in need thereof comprising the steps of: (i)isolating tenocytes from tendon tissue isolated from said subject; (ii)selectively expanding the isolated tenocytes in vitro in a culturemedium comprising: (a) at least 10% v/v serum; (b) about 0.0006% w/vinsulin; and (c) about 0.0002% w/v betamethasone to produce a culture ofexpanded tenocytes comprising at least 80% tenocytes; (iii) seeding abioscaffold with said expanded tenocytes and culturing said bioscaffoldand tenocytes for no more than five days to produce a tenocyte-seededbioscaffold; and (iv) implanting said tenocyte-seeded bioscaffold intothe rotator cuff tear and securing with sutures.
 20. The method of claim19, wherein said bioscaffold comprises a matrix, a membrane, amicrobead, a fleece, a thread, a gel, or mixtures thereof.
 21. Themethod of claim 19, wherein said bioscaffold comprises a type I/IIIcollagen matrix or a small intestinal submucosa.
 22. The method of claim19, wherein the step of seeding a bioscaffold with said expandedtenocytes to produce a tenocyte seeded bioscaffold is carried out at acell density of 0.05 to 5 times the physiological cell density of nativetendon.
 23. The method of claim 22, wherein the cell density is at least1×10⁶ to 1×10⁷ cells per cm².
 24. The method of claim 22, wherein thecell density is 4×10⁶ cells per cm².
 25. The method of claim 19, whereinsaid culture medium comprises at least 15% v/v serum.
 26. A method oftreating a massive rotator cuff tear in a human subject in need thereofcomprising the steps of: (i) isolating tenocytes from tendon tissueisolated from said subject; (ii) selectively expanding the isolatedtenocytes in vitro in a culture medium comprising: (a) at least 15% v/vserum; (b) about 0.0006% w/v insulin; and (c) about 0.0002% w/vbetamethasone to produce a culture of expanded tenocytes comprising atleast 80% tenocytes; (iii) seeding a bioscaffold with said expandedtenocytes to a cell density of 4×10⁶ cells per cm² and culturing saidbioscaffold and tenocytes for no more than five days in said culturemedium to produce a tenocyte-seeded bioscaffold; and (iv) implantingsaid tenocyte-seeded bioscaffold into the rotator cuff tear and securingwith sutures.